The present invention relates generally to stochastic methods for synthesizing random oligomers, with particular emphasis on particle-based synthesis methods. The invention also relates to the use of identification tags on the particles to facilitate identification of the oligomer sequence synthesized. Yet another aspect of the invention relates to the use of tagged oligomer libraries in receptor-binding studies.
The relationship between structure and activity of molecules is a fundamental issue in the study of biological systems. Structure-activity relationships are important in understanding, for example, the function of enzymes, the ways in which cells communicate with each other, and cellular control and feedback systems. Certain macromolecules are known to interact and bind to other molecules having a very specific three-dimensional spatial and electronic distribution. Any large molecule having such specificity can be considered a receptor, whether the molecule is an enzyme catalyzing hydrolysis of a metabolic intermediate, a cell-surface protein mediating membrane transport of ions, a glycoprotein serving to identify a particular cell to its neighbors, an IgG-class antibody circulating in the plasma, an oligonucleotide sequence of DNA in the genome, or the like. The various molecules that receptors selectively bind are known as ligands.
Many assays are available for measuring the binding affinity of known receptors and ligands, but the information that can be gained from such experiments is often limited by the number and type of available ligands. Novel ligands are sometimes discovered by chance or by application of new techniques for the elucidation of molecular structure, including x-ray crystallographic analysis and recombinant genetic techniques for proteins.
Small peptides are an exemplary system for exploring the relationship between structure and function in biology. A peptide is a polymer composed of amino acid monomers. When the twenty naturally occurring amino acids are condensed into polymeric molecules, the resulting polymers form a wide variety of three-dimensional configurations, each resulting from a particular amino acid sequence and solvent condition. The number of possible pentapeptides of the 20 naturally occurring amino adds, for example, is 205 or 3.2 million different peptides. The likelihood that molecules of this size might be useful in receptor-binding studies is supported by epitope analysis studies showing that some antibodies recognize sequences as short as a few amino acids with high specificity. Furthermore, the average molecular weight of amino acids puts small peptides in the size range of many currently useful pharmaceutical products. Of course, larger peptides may be necessary for many purposes, and polypeptides having changes in only a small number of residues may also be useful for such purposes as the analysis of structure-activity relationships.
Pharmaceutical drug discovery is one type of research that relies on studies of structure-activity relationships. In most cases, contemporary pharmaceutical research can be described as the process of discovering novel ligands with desirable patterns of specificity for biologically important receptors. Another example is research to discover new compounds for use in agriculture, such as pesticides and herbicides.
Prior methods of preparing large numbers of different oligomers have been painstakingly slow when used at a scale sufficient to permit effective rational or random screening. For example, the xe2x80x9cMerrifieldxe2x80x9d method (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963), which is incorporated herein by reference) has been used to synthesize peptides on a solid support. In the Merrifield method, an amino add is covalently bonded to a support made of an insoluble polymer. Another amino add with an alpha protected group is reacted with the covalently bonded amino acid to form a dipeptide. The protective group is removed, and a third amino acid with an alpha protective group is added to the dipeptide. This process is continued until a peptide of a desired length and sequence is obtained. Using the Merrifield method, one cannot economically and practically synthesize more than a few peptide sequences in a day.
To synthesize larger numbers of oligomer sequences, others have proposed the use of a series of reaction vessels for oligomer synthesis. For example, a tubular reactor system may be used to synthesize a linear oligomer on a solid phase support by automated sequential addition of reagents. This method still does not enable the synthesis of a sufficiently large number of oligomer sequences for effective economical screening.
Methods of preparing a plurality of oligomer sequences are also known in which a foraminous container encloses a known quantity of reactive solid supports, the solid supports being larger in size than openings of the container. See U.S. Pat. No. 4,631,211, incorporated herein by reference. The containers may be selectively reacted with desired materials to synthesize desired sequences of product molecules. As with other methods known in the art, this method cannot practically be used to synthesize a sufficient variety of polypeptides for effective screening.
Other techniques have also been described. One bead-based method is described in PCT patent publication No. 92/00091, incorporated herein by reference. These methods include the synthesis of peptides on 96 plastic pins that fit the format of standard microtiter plates. See PCT patent publications 84/03564; 86/00991; and 86/06487, each of which is incorporated herein by reference. Unfortunately, while these techniques have been somewhat useful, substantial problems remain. For example, these methods continue to be limited in the diversity of sequences which can be economically synthesized and screened.
Others have developed recombinant methods for preparing collections of oligomers. See PCT patent publication Nos. 91/17271 and 91/19818, each of which is incorporated herein by reference. In another important development, scientists combined the techniques of photolithography, chemistry, and biology to create large collections of oligomers and other compounds on the surface of a substrate. See U.S. Pat. No. 5,143,854 and PCT patent publication Nos. 90/15070 and 92/10092, each of which is incorporated herin by reference.
In the recombinant and VLSIPS(trademark) combinatorial methods, one can uniquely identify each oligomer in the library by determining the coding sequences in the recombinant organism or phage or by the location of the oligomer on the VLSIPS(trademark) chip. In other methods, however, the identity of a particular oligomer may be difficult to ascertain. What is needed in these latter methods is an efficient and simple-to-use method for tagging each particle. Although tagging methods have been developed for large objects, see PCT patent publication Nos. 90/14441 and 87/06383, each of which is incorporated herein by reference, such methods are still needed for combinatorial libraries of oligomers.
From the above, one can recognize that improved methods and apparatus for synthesizing a diverse collection of chemical sequences would be beneficial.
The present invention provides a general stochastic method for synthesizing libraries of random oligomers. The random oligomers are synthesized on solid supports, or particles, but may be cleaved from these supports to provide a soluble library. The oligomers are composed of a sequence of monomers, the monomers being any member of the set of molecules that can be joined together to form an oligomer or polymer, i.e., amino acids, carbamates, sulfones, sulfoxides, nucleosides, carbohydrates, ureas, phosphonates, lipids, esters, combinations of the same, and the like. The library is then screened to isolate individual oligomers that bind to a receptor or possess some desired property. Each oligomer sequence in the library is unique, in a preferred embodiment. In another preferred embodiment, the solid supports are nonporous beads. The solid supports may be composed of a single particle, or two or more linked particles.
A further embodiment of the invention relates to the use of an identifier tag to identify the sequence of monomers in the oligomer. The identifier tag, which may be attached directly to the oligomer with or without an accompanying particle, to a linker attached to the oligomer, to the solid support upon which the oligomer is synthesized, or to a second particle attached to the oligomer-carrying particle, may be any recognizable feature that in some way carries the required information, and that is decipherable at the level of one or a few solid supports. The solid supports may be joined to the oligomers and the identifier tag by means of one or more linker molecules.
In a preferred embodiment, the identifier tag will be an oligonucleotide, preferably composed of pyrimidines or pyrimidines and purine analogs or any type of nucleoside that will not degrade under the coupling conditions used to assemble the oligomer library. The oligonucleotide identifier tag may contain a 5xe2x80x2 and a 3xe2x80x2 amplification site, to allow amplification of the tag by, for example, the polymerase chain reaction (see U.S. Pat. Nos. 4,683,202; and 4,965,188, each of which is incorporated herein by reference). A DNA sequencing primer site, which may be specific for each step of the oligomer synthesis, may also be included in the oligonucleotide tag in addition to the amplification primer sites. The tag may be designed to include, in the oligonucleotide sequence, information allowing identification of the monomer associated with the addition of the particular tag. The oligonucleotide tag will be about 50 to 100 nucleotides in length, in a preferred embodiment.
In another preferred embodiment, the identifier tag may be composed of a set of light-addressable compounds, such as fluorescent or phosphorescent compounds that can be photobleached, which compounds are incorporated into the beads or particles on which the oligomers of the oligomer library are synthesized. Such compounds are widely known in the art.